In order to acquire information useful in diagnosis, stage classification understanding and treatment of human diseases, it is necessary to know the sequences of human protein that are estimated in excess of 30,000 and to identify the important change in expression of protein that announces imminent crisis of the disease. It is also necessary to accurately classify the subtype of the disease on the molecular level so that the function, interrelation and activity of the protein closely related with the process of the disease can be adjusted. One of the bottommost ways to understand the function of the protein is to functionally associate the change of the level of expression with the vegetative stage, cell cycle state, stage of the disease, external stimulation, expression level or any other variable and, although DNA microarray analysis leads to mRNA expression assay method on the genome scale, no direct relation is often found between the in-vivo concentration of mRNA and the coded protein. Accordingly, the difference in speed of translation of mRNA to protein and the difference in speed of in-vivo proteolysis are considered a factor that results in disturbance to the extrapolation of mRNA to the protein expression profile.
Also, such a microarray assay referred to above often plays an important role in protein functional regulation, but is unable to detect, identify or quantitatively determine the protein modification.
Accordingly, the quantitative analysis against the detection and the analysis of analyte of a low concentration contained various biological samples generally necessitates labelling with the use of a radioactive isotope or a fluorescence reagent, and such method generally requires a substantial amount of time and is, hence, inconvenient to accomplish. For example, although various qualitative analyzing methods, two dimensional electrophoretic method and liquid chromatography have been widely used for the protein profiling, they are not adequate for use in summary survey.
Yet, in view of the solid state sensor, particularly biosensor, that is increasingly used in chemical, biological or pharmaceutical studies, such sensor is in recent years drawing unprecedented attention and makes use of a conversion structure capable of converting two elements; a recognition element of high uniqueness and a molecular recognition event, into a quantifiable signal and has been developed with the aim of detecting various biological molecular complexes including oligonucleotide pairs, antibody-antigen complex, hormone-acceptor complex, enzyme-substrate complex and lectin-glycoprotein complex interaction, but it still insufficient.
In view of the foregoing, the use of Raman scattering spectroscopy or a surface plasmon resonance has been suggested with the aim of accomplishing an objective to enable a highly accurate detection or identification of individual molecules in the biological sample. The wavelength of Raman scattering spectroscopy is characteristic of a chemical composition and structure of Raman scattering molecules in the sample and the intensity of Raman scattered light depends on the molecular concentration in the sample. In the practice of Raman scattering spectroscopy, nanoparticles of gold, silver, copper and any other plasmon metal exhibit a surface intensified Raman scattered effect in response to the applied laser beams and, using it, biological molecules of interest are characterized such that nucleotide, deoxyadenosine monophosphate, protein and hemoglobin could be detected at a single molecular level. As a result, however, SERS (surface enhanced Raman spectroscopy) could not be considered suitable for use in quantitative analysis of the protein content in the complex biological sample such as blood plasma.
In view of the foregoing, the need has arisen of a method of analyzing the protein composition of a sample of the complex organism in blood serum or the like with the use of Raman scattering spectroscopy to detect or identify the individual proteins with reliability, as well as high throughput means for quantitatively and qualitatively detecting the protein of a low concentration level in a composite sample. Accordingly, a method for analyzing the protein content in a biological sample has been suggested, in the patent document 1 listed below, which method includes isolating protein and protein segments in the sample based on chemical and/or physical characteristics of the protein, maintaining in an isolated condition the isolated proteins at the discrete positions on a solid substrate or in the flow of liquid then flowing, detecting Raman spectrum formed by the isolated proteins at the discrete positions so that through the spectrum at the discrete positions, information on the structure of one or more particular proteins at discrete positions can be provided. Also, SERS phenomenon involves some problems to be resolved in terms of repeatability and reliability because of (1) the mechanism not yet comprehended impeccably, (2) difficulties in formation of the nano-material, which is accurately and structurally defined, and in control, and (3) change in enhancement efficiency brought about by wavelength of light used in measurement of the spectrum and direction of polarization and, therefore, application of SERS including development and commercialization of the nano-biosensor is considerably affected. For this reason, a technique has been suggested, in the patent document 2 listed below, in which a hybrid structure of nanowire and nanoparticles is utilized to enhance SERS signals of such biomolecular as bio-extract and protein, DNA, repeatability of measurement and increase of sensitivity and reliability.